Phenylalanine Ammonia-Lyase from Loblolly Pine

Abstract

Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5' end of the clone isolated from the expression library, was also used to isolate cDNA clones. These methods yielded cDNA clones covering the protein coding region of the pal messenger RNA. Comparisons of nucleotide sequence of pal cDNAs from pine, bean, sweet potato, and rice showed 60 to 62% identity between the pine clone and the angiosperm clones.The development of secondary xylem in woody perennials is an interesting biological process that has received little research attention at the molecular level. Inquiry into the processes of wood differentiation has been limited to physiological and ultrastructural studies, which have laid a foundation upon which to build a more detailed understanding of intracellular events. We are beginning to apply tools of molecular biology to problems of xylem differentiation and development that are difficult to address at the level of physiology and ultrastructure.We have chosen to focus on lignification as an important aspect of wood formation. Lignin is a complex polymer of three phenylpropanoid subunits deposited in the cell walls of vascular elements after cell growth has ceased. Sucrose supplied by the phloem is transformed into phenylalanine via the shikimic acid pathway, and lignin monomers are produced from phenylalanine by a branch of phenylpropanoid metabolism. The pathway of lignin precursor biosynthesis is well established (see ref. 7 for review). The first step of the phenylpropanoid pathway is deamination of phenylalanine by ' Supported by the North Carolina State University Forest Biotechnology Industrial Associates Group. PAL2 (EC 4.3.1.5) to yield trans-cinnamic acid. Hydroxylation of cinnamic acid produces p-coumaric acid; additional hydroxylation and methylation of p-coumarate produce first ferulic acid, then sinapic acid. These three acids are coupled to CoA and reduced to the corresponding alcohols. The end products of this pathway are p-coumaryl, coniferyl, and sinapyl alcohols. The relative proportions of the three precursors in lignin varies from species to species and even between cell types within a single individual (14). In general, lignin in angiosperms contains more p-coumaryl and sinapyl alcohol residues than does gymnosperm lignin; coniferyl alcohol is the primary lignin monomer in most gymnosperms.The regulation of ...

Expression of three phenylpropanoid pathway genes in Scots pine (Pinus sylvestris L.) in open-pollinated families with differing relative wood densities during early and late wood formation

“…The PAL1, CCR and CAD genes were selected for this study because of their previously described importance in wood formation in other species (WHETTEN and SEDEROFF, 1992;WHETTEN and SEDEROFF, 1995;LI et al, 2006;O'MALLEY et al, 1992). For the PAL1 gene, a P. sylvestris gene sequence was available, while for the other two genes, sequences from P. taeda were utilised for primer design.…”

“…In Pinus taeda, a single PAL gene was identified in a xylem-specific cDNA library (WHETTEN and SEDEROFF, 1992), while in angiosperm genomes, PAL belongs to a multigene family, and it has been suggested that gymnosperm and angiosperm lignin biosynthesis pathways are highly distinct. However, in Pinus banksiana, research suggests that PAL is encoded by a multigene family (BUTLAND et al, 1998), and it has been reported recently that there are actually five PAL genes in the P. taeda genome (BAGAL et al, 2012).…”

Biochemical Evaluation of Phenylalanine Ammonia Lyase (PAL) from Endemic Cyathobasis fruticulosa (Bunge) Aellen. for the Dietary Treatment of Phenylketonuria (PKU)

“…The enzyme was partially purifi ed in a two-step procedure involving ammonium sulfate (Merck, Darmstadt) precipitation and dialysis (21,22). The enzyme extract was brought to 60 % saturation by gradual addition of solid ammonium sulfate while stirring at 4 °C.…”

Rhizosphere and endophytic bacteria for induction of salt tolerance in gladiolus grown in sodic soils

“…Polyphenol oxidase (PPO) was assayed using the modified method of Mayer et al (1966) using 0.1 M PB (pH 6.5) and 0.01 N catechol along with enzyme extract. The phenyl alanine lyase (PAL) assay was conducted using 400 ml of 50 mM Tris-HCl (pH 8.8) and 600 ml of 1 mM Lphenylalanine along with enzyme extract as reaction mixture (Whetten & Sederoff 1992).…”